Collagen comprises one-third of the protein in humans. Collagen abnormalities are associated with a wide variety of human diseases, such as osteogenesis imperfecta, Ehlers-Danlos syndrome, and some types of osteoporosis and arthritis. The long-term objective of the proposed research is to reveal in atomic detail the chemical basis for the unique triple-helical structure of collagen, and the roles of collagen in human health and disease. Specific Aims: (1) Basis for Triple Helix Stability. The hypothesis to be evaluated is that individual strands of collagen are preorganized to form a triple helix. The experiments make use of collagen strands containing nonnatural amino acids (in particular, 4-fluoroproline diastereomers and N-methylglycine) that impose distinct stereoelectronic and steric effects. Data on conformational stability will be reinforced by computational and structural analyses. (2) Cystine Knot Templates. A collagen triple helix cross-linked by two disulfide bonds (that is, a cystine knot) can contain three different strands, and provides a realistic mimic of natural collagen. An efficient solid-phase synthesis of triple helices cross-linked by a cystine knot will be developed, and those helices will be used to evaluate the contribution of the ladder of interstrand main-chain-main-chain hydrogen bonds to collagen stability, assess the ability of a single strand of collagen to invade a triple helix, and self-assemble long, well-defined triple helices. (3) Encoded Collagen Library. Phage display of a cystine knot will be used to generate encoded libraries of collagen triple helices. These libraries will be screened for triple helices with high affinity for collagen-binding proteins, in particular, the adhesin protein from the pathogenic bacterium Staphyloccocus aureus. Significance: The results of the proposed research will provide new insights into the structure and conformational stability of the collagen triple helix and on its interaction with other proteins, and could ultimatley lead to the creation of collagen mimics and collagen-based biomaterials with important therapeutic applications.